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Topalov N.N.,Center for Theoretical Problems of Physicochemical Pharmacology | Yakimenko A.O.,HemaCore Ltd. | Canault M.,Aix - Marseille University | Canault M.,French Institute of Health and Medical Research | And 13 more authors.
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2012

OBJECTIVE-: Phosphatidylserine (PS) externalization by platelets upon activation is a key event in hemostasis and thrombosis. It is currently believed that strong stimulation of platelets forms 2 subpopulations, only 1 of which expresses PS. METHODS AND RESULTS-: Here, we demonstrate that physiological stimulation leads to the formation of not 1 but 2 types of PS-expressing activated platelets, with dramatically different properties. One subpopulation sustained increased calcium level after activation, whereas another returned to the basal low-calcium state. High-calcium PS-positive platelets had smaller size, high surface density of fibrin(ogen), no active integrin αIIbβ3, depolarized mitochondrial membranes, gradually lost cytoplasmic membrane integrity, and were poorly aggregated. In contrast, the low-calcium PS-positive platelets had normal size, retained mitochondrial membrane potential and cytoplasmic membrane integrity, and combined retention of fibrin(ogen) with active αIIbβ 3 and high proaggregatory function. Formation of low-calcium PS-positive platelets was promoted by platelet concentration increase or shaking and was decreased by integrin αIIbβ3 antagonists, platelet dilution, or in platelets from kindlin-3-deficient and Glanzmann thrombasthenia patients. CONCLUSION-: Identification of a novel PS-expressing platelet subpopulation with low calcium regulated by integrin αIIbβ3 can be important for understanding the mechanisms of PS exposure and thrombus formation. © 2012 American Heart Association, Inc.

Deppermann C.,University of Wurzburg | Cherpokova D.,University of Wurzburg | Nurden P.,University of Wurzburg | Schulz J.-N.,University of Cologne | And 10 more authors.
Journal of Clinical Investigation | Year: 2013

Platelets are anuclear organelle-rich cell fragments derived from bone marrow megakaryocytes (MKs) that safeguard vascular integrity. The major platelet organelles, α-granules, release proteins that participate in thrombus formation and hemostasis. Proteins stored in α-granules are also thought to play a role in inflammation and wound healing, but their functional significance in vivo is unknown. Mutations in NBEAL2 have been linked to gray platelet syndrome (GPS), a rare bleeding disorder characterized by macrothrombocytopenia, with platelets lacking α-granules. Here we show that Nbeal2-knockout mice display the characteristics of human GPS, with defective α-granule biogenesis in MKs and their absence from platelets. Nbeal2 deficiency did not affect MK differentiation and proplatelet formation in vitro or platelet life span in vivo. Nbeal2-deficient platelets displayed impaired adhesion, aggregation, and coagulant activity ex vivo that translated into defective arterial thrombus formation and protection from thrombo-inflammatory brain infarction following focal cerebral ischemia. In a model of excisional skin wound repair, Nbeal2-deficient mice exhibited impaired development of functional granulation tissue due to severely reduced differentiation of myofibroblasts in the absence of α-granule secretion. This study demonstrates that platelet α-granule constituents are critically required not only for hemostasis but also thrombosis, acute thrombo-inflammatory disease states, and tissue reconstitution after injury.

Fang J.,Medical College of Wisconsin | Fang J.,Childrens Research Institute | Fang J.,Fund Research Center | Nurden P.,Plateforme Technologique et dInnovation Biomedicale | And 11 more authors.
Journal of Thrombosis and Haemostasis | Year: 2013

Background and objectives: β3-Deficient megakaryocytes were modified by human β3-lentivirus transduction and transplantation to express sufficient levels of a C560Rβ3 amino acid substitution, for investigation of how an activated αIIbβ3 conformation affects platelets in vivo in mice. Patient/Methods: As in our previous report of an R560β3 mutation in a patient with Glanzmann thrombasthenia, R560β3 murine platelets spontaneously bound antibody that only recognizes activated αIIbβ3 bound to its ligand, fibrinogen. Results: With this murine model, we showed that αIIb-R560β3 mutation-mediated continuous binding of fibrinogen occurred in the absence of P-selectin surface expression, indicating that the integrin was in an active conformation, although the platelets circulated in a quiescent manner. Remarkably, only 35% of R560β3 'mutant' mice survived for 6 months after transplantation, whereas 87% of C560β3 'wild-type' mice remained alive. Pathologic examination revealed that R560β3 mice had enlarged spleens with extramedullary hematopoiesis and increased hemosiderin, indicating hemorrhage. R560β3 megakaryocytes and platelets showed abnormal morphology and irregular granule distribution. Interestingly, R560β3 washed platelets could aggregate upon simultaneous addition of fibrinogen and physiologic agonists, but aggregation failed when platelets were exposed to fibrinogen before activation in vitro and in vivo. Conclusions: The results demonstrate that continuous occupancy of αIIbβ3 with fibrinogen disrupts platelet structure and function, leading to hemorrhagic death consistent with Glanzmann thrombasthenia rather than a thrombotic state. © 2013 International Society on Thrombosis and Haemostasis.

Laguerre M.,CNRS Institute of Chemistry | Sabi E.,University of Sheffield | Daly M.,University of Sheffield | Stockley J.,University of Sheffield | And 4 more authors.
PLoS ONE | Year: 2013

Mutations in ITGA2B and ITGB3 cause Glanzmann thrombasthenia, an inherited bleeding disorder in which platelets fail to aggregate when stimulated. Whereas an absence of expression or qualitative defects of αIIbβ3 mainly affect platelets and megakaryocytes, αvβ3 has a widespread tissue distribution. Little is known of how amino acid substitutions of β3 comparatively affect the expression and structure of both integrins. We now report computer modelling including molecular dynamics simulations of extracellular head domains of αIIbβ3 and αvβ3 to determine the role of a novel β3 Pro189Ser (P163S in the mature protein) substitution that abrogates αIIbβ3 expression in platelets while allowing synthesis of αvβ3. Transfection of wild-type and mutated integrins in CHO cells confirmed that only αvβ3 surface expression was maintained. Modeling initially confirmed that replacement of αIIb by αv in the dimer results in a significant decrease in surface contacts at the subunit interface. For αIIbβ3, the presence of β3S163 specifically displaces an α-helix starting at position 259 and interacting with β3R261 while there is a moderate 11% increase in intra-subunit H-bonds and a very weak decrease in the global H-bond network. In contrast, for αvβ3, S163 has different effects with β3R261 coming deeper into the propeller with a 43% increase in intra-subunit H-bonds but with little effect on the global H-bond network. Compared to the WT integrins, the P163S mutation induces a small increase in the inter-subunit fluctuations for αIIbβ3 but a more rigid structure for αvβ3. Overall, this mutation stabilizes αvβ3 despite preventing αIIbβ3 expression. © 2013 Laguerre et al.

Nurden A.T.,Plateforme Technologique et dInnovation Biomedicale | Pillois X.,Plateforme Technologique et dInnovation Biomedicale | Pillois X.,French Institute of Health and Medical Research | Wilcox D.A.,Blood Research Institute | Wilcox D.A.,Medical College of Wisconsin
Seminars in Thrombosis and Hemostasis | Year: 2013

Glanzmann thrombasthenia (GT) is the principal inherited disease of platelets and the most commonly encountered disorder of an integrin. GT is characterized by spontaneous mucocutaneous bleeding and an exaggerated response to trauma caused by platelets that fail to aggregate when stimulated by physiologic agonists. GT is caused by quantitative or qualitative deficiencies of αIIbβ3, an integrin coded by the ITGA2B and ITGB3 genes and which by binding fibrinogen and other adhesive proteins joins platelets together in the aggregate. Widespread genotyping has revealed that mutations spread across both genes, yet the reason for the extensive variation in both the severity and intensity of bleeding between affected individuals remains poorly understood. Furthermore, although genetic defects of ITGB3 affect other tissues with β3 present as αvβ 3 (the vitronectin receptor), the bleeding phenotype continues to dominate. Here, we look in detail at mutations that affect (i) the β-propeller region of the αIIb head domain and (ii) the membrane proximal disulfide-rich epidermal growth factor (EGF) domains of β3 and which often result in spontaneous integrin activation. We also examine deep vein thrombosis as an unexpected complication of GT and look at curative procedures for the diseases, including allogeneic stem cell transfer and the potential for gene therapy. © 2013 by Thieme MedicalPublishers, Inc.

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